A Comparison of Single-Sized and Mixed-Sized Particles on Lateral Flow Assay Performance for SARS-CoV-2 Nucleocapsid Protein Detection

There are several factors that can affect the sensitivity and reproducibility of the lateral flow assay, such as the size of gold nanoparticles (GNPs) employed. This study evaluated the analytical performance of single-sized and mixed-sized GNPs for conjugation with detection antibodies of the lateral flow assay (LFA) platform. This platform was used as a model assay to diagnose SARS-CoV-2 nucleocapsid protein (N protein) levels and demonstrate the analytical performance of single-sized and mixed-sized GNPs in LFA tests. Two sizes of GNP (40 nm and 80 nm) were used for conjugating detection antibodies (GNPs@anti-SARS-CoV-2 N protein antibody) that were then mixed at different ratios. The unique optical properties of the GNPs induced a distinguishing color-shedding effect on the single- and mixed-sized GNPs@anti-SARS-CoV-2 N protein antibody conjugates interacting with the target analyte N-protein spotted on the test line. Using mixed-sized GNPs@anti-SARS-CoV-2 N protein antibody conjugates enhanced the signal 2-fold relative to the 40 nm GNPs, providing superior stability compared to solely employing the large GNPs (80 nm). The proposed platform of multiple-mixed-sized GNPs used in this study could provide an efficient SARS-CoV-2 N protein detection mechanism that can be utilized as a model biomarker for early acute respiratory distress syndrome (ARDS) detection, which can detect the signal at 50 ng/ml.